Erythropoietin (EPO) is a glycoprotein which induces production of erythrocytes in the bone marrow by stimulating differentiation of erythroid progenitor cells. EPO consists of 165 amino acids. After purification of erythropoietin from human urine by Mijake in 1977, it has become possible to produce large amounts by genetic engineering technology. Erythropoietin was found to be able to induce effectively various hematopoiesis in the treatment of anemia resulting from a chronic renal insufficiency and various types of anemia by several causes, and in use during certain surgical procedures (Mijake et al., J. Biol. Chem. 25, 5558-5564, 1977; Eschbach et al., New Engl. J. Med. 316, 73-78, 1987; Sandford. B. K, Blood, 177, 419-434, 1991; WO 85-02610). For this reason, erythropoietin has been used as a pharmaceutical in various indications of diseases for a long time. However, like other protein pharmaceuticals, erythropoietin protein should also be carefully prepared to prevent denaturation from being caused by the loss of stability, for the purpose of the effective usage thereof.
Generally, proteins have a short half-life and denaturation easily occurs such as by aggregation of monomers, precipitation by aggregation, and adsorption to ampoule walls when exposed to extreme temperatures, interface of water and air, high pressure, physical and mechanical stresses, organic solvents, contamination by microorganisms and the like. Denatured proteins lose their native physiochemical properties and physiological activity, and the denaturation of protein is generally irreversible. Thus, proteins cannot recover their native properties, once denatured. Especially, in case of proteins such as erythropoietin, which is administered in single dosages as small as a few micrograms, when they are adsorbed to the ampoule wall due to the disappearance of stability, the loss resulting therefrom is relatively considerable. Furthermore, the protein thus adsorbed easily aggregates via a denaturation process, and administration of the denatured protein causes antibodies, as spontaneously produced proteins, to be formed against this denatured protein in a body, thus the protein should be administered in the substantially stable form. Accordingly, various methods to prevent protein denaturation in aqueous solution have been studied (John Geigert, J. Parenteral Sci. Tech., 43, No5, 220-224, 1989; David Wong, Pharm. Tech. October, 34-48, 1997; Wei Wang, Int. J. Pharm., 1 85, 129-188, 1999; Willem Norde, Adv. Colloid Interface Sci., 25, 267-340, 1986; Michelle et al., Int. J. Pharm. 120, 179-188, 1995).
Some protein formulations solved the denaturation with a lyophilization method. However, lyophilized products are inconvenient since they have to be reconstituted prior to injection, and a large capacity freeze-dryer is required for processing, therefore, extensive investment is necessary. A method of producing powdered forms of protein using spray-drying techniques is also used; however, it has disadvantages in that the economic efficiency decreases due to low yield and exposure to high temperature can cause protein denaturation during the process.
As an alternative way to solve the limitation of the above methods, there is a method to improve the protein stability by adding stabilizers to an aqueous protein solution. As protein stabilizers, there are known surfactants, serum albumin, polysaccharides, amino acids, macromolecules and salts (John Geigert, J. Parenteral Sci. Tech., 43, No. 5, 220-224, 1989; David Wong, Pharm. Tech., October, 34-48, 1997; Wei Wang., Int. J. Pharm., 185, 129-188, 1999). However, suitable stabilizers should be selected in accordance with physiochemical characteristics of each protein; otherwise, for example, when stabilizers are used in certain combinations, competitive reaction or side reaction can occur to result in negative effects different from the intended effects. Moreover, since an appropriate range of concentrations exists for each stabilizer, much effort and caution are needed to stabilize aqueous proteins (Wei Wang, Int. J. Pharm., 185, 129-188, 1999).
Among the protein stabilizers, serum albumin and gelatin, derived from human or animal, are generally used as stabilizers of aqueous protein formulations and have been proven to be effective. However, there is a risk of viral contamination with human-derived serum albumin, and gelatin and bovine serum albumin may transmit diseases like “Transmissible Spongiform Encephalopathies”, or raise allergies in some patients; therefore, in Europe, use of materials from human and animal sources as pharmaceutical additives is increasingly restricted (EMEA/CPMP/BWP/450/01 Report from the Expert Workshop on Human TSEs and Medicinal products derived from Human Blood and Plasma (1 Dec. 2000), CPMP/PS/201/98 Position Statement on New Variant CJD and Plasma-Derived Medicinal Products (Superseded by CPMP/BWP/2879/02). Thus, it is necessary to develop methods of formulating stable protein formulations without serum albumin from human or animal, solving the problems of existing erythropoietin formulations containing serum albumin.
In U.S. Pat. No. 4,879,272, there is disclosed the addition of human/bovine serum albumin, lecithin, dextran and cellulose as agents to inhibit protein adherence to ampoule walls. According to this patent, the recovery yield of erythropoietin is good at 69˜98% after storage for about 2 hours at 20° C., compared with only 16% without such an addition, but it has a problem that the loss due to adsorption can be considerable.
In U.S. Pat. No. 4,806,524, there are disclosed the lyophilized formulation and aqueous formulation of erythropoietin, using polyethylene glycol, protein, saccharides, amino acids, organic salts and inorganic salts as stabilizers for erythropoietin. According to this patent, after storage of about 7 days at 25° C., the lyophilized formulation has a high recovery yield level of 87˜98%, but the aqueous formulation has a low recovery yield level of only 60˜70%, thus the aqueous formulation is relatively less stable.
In U.S. Pat. No. 4,992,419, there are disclosed the aqueous formulation and lyophilized formulation of erythropoietin, in which 0.5˜5 g/L of non-ionic surfactant as an anti-absorption agent, and 5˜50 g/L of urea and 5˜25 g/L of amino acids as stabilizers were used. However, this patent has problems that the aqueous formulation shows a limited stability compared to the formulations containing human serum albumin, and the lyophilized formulation requires the reconstitution process so as to maintain a sufficient activity.
In U.S. Pat. No. 5,376,632, there are disclosed the aqueous formulation, lyophilized formulation and spray-dried powder formulation of erythropoietin, containing β or γ cyclodextrins but not containing other additional pharmaceutical excipient. However, the formations using cyclodextrins are not practical due to their renal toxicity.
In U.S. Pat. No. 5,661,125, there are disclosed the formation of erythropoietin, containing benzyl alcohol, parabens, phenol and mixtures thereof, and an experiment showing the stability thereof compared to the formation of erythropoietin containing human serum albumin. However, the formulation of this patent showed a low stability and significant precipitation of erythropoietin even at low temperature.
In WO 01/87329 A1, there are disclosed the aqueous formulations of erythropoietin and a multiple charged inorganic anion in a pharmaceutically acceptable buffer to keep the solution pH in the range from about pH 5.5 to about pH 7.0. This application shows the comparative experiment regarding stability in which, after storage of EPO and PEGylated EPO at various temperatures for 6 months, the content of sialic acid and the standard bioactivity (%) of each EPO were measured in various formulations; however, since the amount of EPO monomers was not measured, the recovery yield (%) of EPO monomers cannot be precisely determined.
Therefore, it is desired to provide the new aqueous formulation that has a long-term stability without using protein components derived from animal such as serum albumin.